The antifungal hexapeptidolactones of the kutzneride family are most probably products of nonribosomal peptide synthetase assembly lines. These macrolactones are of special interest since all six of the residues derive from unusual nonproteinogenic monomers, three of which had not been reported before, suggesting novel enzyme chemistry. The first goal of this proposed project is to clone the kutzneride biosynthetic cluster from Kutzneria genomic DNA and perform functional characterization of amino acids activated by the adenylation domains to confirm that the kutzneride cluster had indeed been cloned. The second goai is to elucidate the timing and mechanism of the kutzneride enzymatic assembly process, which should reveal some of the molecular logic for how this antifungal depsipeptide is produced, including the timing of enzymatic generation of all six unusual building blocks. Understanding the enzymatic machinery to make novel building blocks and when they are tailored in nascent product scaffolds will be of high value to establish their portability to other assembly lines. The ability to reprogram those enzyme cascades will increase the probability of success to make novel structures with improved therapeutic properties. Relevance: Natural products continue to provide important scaffolds for new anticancer drugs. Deciphering the logic, machinery and mechanism of natural product biosynthetic pathways provides context for re-engineering these pathways and may lead to the discoveries of molecules with novel mechanisms of therapeutic action. The antifungal hexapeptidolactones of the kutzneride family derive from unusual nonproteinogenic monomers and understanding the enzymatic machinery to make these novel building blocks and when they are tailored in nascent product scaffolds will aid efforts to create collections of natural product varients with novel structures and improved therapeutic properties.